Technical Abstract:
Bovine mastitis results in annual losses between $1.7 billion and $2 billion in the United States alone. Among the most relevant causative agents of this disease are Streptococcus agalactiae (Group B; GBS) and Streptococcus dysgalactiae (Group C; GCS) streptococci as well as Staphylococcus aureus. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a new promising source of antimicrobials against these pathogens. In this work, we biochemically characterized the endolysins of the streptococcal phages B30 and 'SA2, examining the influence of salt concentration, pH, and bivalent metal cations on their in vitro lytic activity against S. dysgalactiae. When used in combination, the B30 and 'SA2 lysins were found to exhibit a synergistic effect against streptococci. Using a plate lysis checkerboard assay, the sum of the fractional inhibitory concentrations of both enzymes against S. dysgalactiae was determined to be 0.5, indicating strong synergy. Furthermore, we examined the activity of both lysins against different streptococcal species in whole milk. The addition of 100 µg/ml B30 or 'SA2 lysin to milk samples inoculated with 3 x 103 cfu/ml instantaneously reduced the cell concentrations and kept them ~2 log units (for B30) or ~3 log units (for 'SA2) below those of the control samples during an incubation period of three hours at 37°C. Finally, we created fusion proteins consisting of the 'SA2 endopeptidase domain and two different SH3b cell wall binding domains from the staphylococcal peptidoglycan hydrolases Lysostaphin and LysK. The addition of these SH3b domains resulted in an approximately five-fold increase in lytic activity of these proteins against Staphylococcus aureus cells, as determined by turbidity reduction assays, while still maintaining significant streptolytic activity. When added to whole milk spiked with Staphylococcus aureus cells, 100 µg/ml of 'SA2E-SH3b(Lyso) or 'SA2E-SH3b(LysK) caused a reduction in cell numbers of >1 or 3 log units, respectively. Overall, our results with the B30 and 'SA2 lysins demonstrate the high potential of these enzymes as sources of antimicrobials for the treatment of bovine mastitis.